Martin Jinek

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a spliceosome-independent pathway in which the tRNA splicing endonuclease complex catalyzes intron excision. The resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC), comprising Ashwin, CGI-99, FAM98B, DDX1, and RTCB/HSPC117. The molecular architecture and functions of its non-catalytic subunits remain poorly understood. Using cryo-EM, we determined an atomic-resolution structure of human tRNA-LC. CGI-99, DDX1, and FAM98B form an α-helical bundle that contacts RTCB opposite its active site and anchors DDX1 via its C-terminal helix. FAM98B and CGI-99 form an extensively co-folded heterodimer that clamps Ashwin in a pincer-like structure. Structure-based mutagenesis supports the architecture of the complex. We further show that FAM98A and FAM98C assemble distinct RTCB-containing complexes lacking Ashwin, suggesting specialized cellular functions. Our results provide insights into the molecular assembly of the tRNA ligase complex, highlighting its functions in tRNA biogenesis and beyond.

The tRNA ligase complex (tRNA-LC) seals tRNA exon halves in the nucleus during pre-tRNA splicing and XBP1 -mRNA exons in the cytoplasm as part of the unfolded protein response (UPR). This dual function requires the tRNA-LC to be either nuclear or cytoplasmic. Here, we reveal that Ashwin (ASW), the vertebrate-specific subunit of the tRNA-LC, serves as its nuclear import factor. ASW contains a dual nuclear localisation signal (NLS) which, upon disruption, leads to the retention of the tRNA-LC in the cytoplasm, impairing pre-tRNA splicing with the consequent accumulation of 5’ tRNA fragments. We also show that the tRNA-LC exists in three forms, depending on which FAM98 paralog is bound, either FAM98A, FAM98B or FAM98C. ASW interacts exclusively with the FAM98B-containing complex, ensuring its nuclear localization for tRNA biogenesis. Attaching an NLS to RTCB, the catalytic and indispensable tRNA-LC subunit, rescues pre-tRNA splicing in cells depleted of ASW. We hypothesize that vertebrates evolved ASW to localize a sub-population of tRNA-LC to the nucleus, while using FAM98 paralogs to retain a fraction of RTCB in the cytoplasm to splice XBP1 -mRNA during UPR.

CRISPR-associated transposons (CASTs) are Tn7-like elements that have co-opted RNA-guided CRISPR effectors for targeted DNA insertion. CASTs have been adapted as genome editing tools for programmable, site-specific integration. Among them, the type I-F system from Ps e udoalteromonas ( Pse CAST) shows exceptionally robust activity in human cells, yet its mechanistic basis remains poorly understood. Here, we present structural and biochemical analysis of the Pse CAST transposase TnsAB. Biochemical reconstitution of transposon DNA excision defines key characteristics of the transposition mechanism. Cryogenic electron microscopy (cryo-EM) structures of Pse TnsAB paired-end complexes reveal molecular determinants of transpososome assembly, transposon end recognition and cleavage. We validate these findings using biochemical and in vivo assays of structure-based transposase mutants, and provide mechanistic insights into the enhanced activity of a laboratory-evolved TnsAB variant. Together, our studies highlight molecular features underlying the efficiency of natural and engineered type I-F transposases and establish a mechanistic framework for their continued rational optimization.

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex ^1,2 . Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools ^3–6 . The type I-F Pseudoalteromonas CAST ( Pse CAST) displays the highest activity in mammalian cells to date ⁷ and has been the subject of extensive directed evolution ⁸ , but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation ⁹ . Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the Pse CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa Pse CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

Innate immune systems detect molecular signatures of infection to initiate antiviral defence ^1–3 , yet the identity of pathogen-associated signals that distinguish phage from host nucleic acids remains incompletely understood. While recent work has shown that nucleic acid structures can act as triggers for bacterial defense systems ^4–7 , how these structural signals are coupled with immune activation remain unclear. Here we show that forked DNA structures activate a helicase-nuclease immune complex in type III Druantia through a processing-dependent mechanism. Using cryo-electron microscopy and biochemical reconstitution, we find that the exonuclease DruH processes 3′ DNA termini to generate 5′ overhangs that recruit and activate the helicase-nuclease DruE at duplex-single-stranded DNA junctions. Structural analysis of the DruE-DruH complex reveals how substrate-dependent assembly remodels an autoinhibited helicase dimer into an active DNA degradation complex. Functional assays demonstrate that coordinated nuclease and helicase activities enable efficient degradation of forked DNA substrates and mediate phage defense without detectable host toxicity. Together, our findings define a mechanism in which enzymatic processing of replication-associated DNA structures licenses immune activation, providing a framework for how nucleic acid architecture is coupled to effector activation in bacterial immunity.